Introduction
Blood is of prime importance in the normal physiologic function of our major organ
systems. In order for it to be effective, blood must be in a liquid or non-coagulated
state. Another important function of blood is to maintain an intact circulatory system
following trauma. The process by which blood is maintained fluid within the vessel walls
and the ability of the system to prevent excessive blood loss upon injury is termed
hemostasis. The balance between the forces that cause blood to solidify or to remain fluid
is very delicate and involves several interacting systems.

When you cut yourself,the process of coagulation begins by the formation of a blood
clot. This is followed shortly after by digestion or breakdown of the clot. Patients clot
and/or bleed because of a variety of identifiable hemostatic abnormalities. Logical and
effective treatment depends upon the proper identification of the abnormality. The
coagulation or hemostasis laboratory performs tests to determine the cause and to monitor
the proper treatment of the defect.

This educational module will teach you a systematic and practical approach to
understanding the laboratory's role in diagnosis and therapy of bleeding and clotting
disorders.

Platelets, Vascular and
Clotting Factorsa brief review of their function

Objectives:1. Describe the role platelets play in normal hemostasis.2. Describe hemostasis and the various factors involved with coagulation.

Glossary:

Platelets -

small, anuclear cytoplasmic disks. In an
unstimulated state, the shape is discoid.

Hemostasis -

the process in circulation where the blood is
maintained fluid in vessels and without major loss in case of injury.

Coagulation factors -

Components that exist in the circulation and supply the necessary constituents for clot
formation.

Hemostasis:
The property of the circulation where the circulating fluid is maintained within the blood
vessels is referred to as hemostasis. The process depends on a delicate and complex
interplay of at least 4 systems: vascular, plasma coagulation factors, platelets and
fibrinolytic system.

Vascular System:
Blood normally flows smoothly through the vascular system without cellular adherence to
the vessel wall. The thin layer of endothelial cells lining the inner surface of the
various vessels helps to maintain a thrombo-resistant surface. When vascular injury occurs
following trauma or in certain vessel diseases, the endothelial cells interact with
platelets and clotting factors to form a blood clot at the site of injury.

Platelets and Hemostasis:
The platelet has at least a fourfold function: (1) In response to vascular injury,
platelets are stimulated to initiate the formation of a primary hemostatic plug, (2)
the platelet contributes phospholipid (sometimes referred to as platelet factor 3 or PF3)
to the coagulation cascade, (3) they help maintain vascular integrity through
endothelial support and (4) platelets may have a role in inflammatory response,
possibly by activating the fifth component of complement.

There is a sequence of events which occurs at the site of vascular injury. First, the
platelet is attracted to the exposed sub-endothelial layer of collagen and adheres to it.
To accomplish this, the platelet undergoes a shape change. Secondly, the platelets release
intrinsic adenosine diphosphate (ADP), among other substances. The released ADP stimulates
other platelets to stick together at the wound site, and, thirdly, aggregation occurs. In
this process, platelets adhere to each other to form a beginning plug. Finally,
coagulation occurs and fibrin forms around the platelet aggregate to initiate repair.(See figure 1)

Coagulation Factors:
The coagulation factors circulate in the plasma as cofactors or as procoagulants, and,
when activated supply some of the components needed for clot formation. According to the
international nomenclature system, coagulation cofactors and procoagulants were assigned
roman numerals in the order of their discovery and don't correspond to their location in
the coagulation sequence of activation. (See figure 2) The
coagulation factors are generated in the liver cells, except for Factor VIII (at least the
Von Willebrand's portion), which is produced in multiple organs, possibly the endothelial
cells and megakarocytes.

The model generally used to describe the mechanism of coagulation is the cascade
system. The cascade is separated into three areas: the intrinsic system, commonly measured
by the aPTT test, which is activated by surface contact; the extrinsic system, commonly
measured by the PT test, which is activated by vascular injury, and, the common pathway,
which is set into motion by activation from the intrinsic and/or the extrinsic pathway.
Because of the variety of constituents involved with the common pathway, there are several
different tests that could be used to monitor activity. The systems and tests are
described in later sections of this module.

Primary Hemostasis:
Following injury to a blood vessel, all of the systems are activated. For sake of ease,
the hemostatic process is divided into 2 components; primary hemostasis and secondary
hemostasis. Primary hemostasis depends upon the response of the platelet and blood vessel
wall to the injury. When the small blood vessels are injured, blood platelets adhere and
aggregate at the site of injury, reducing and finally arresting bleeding.

Secondary Hemostasis:
Secondary Hemostasis starts when the cascade system of Coagulation is activated by
substances released at the time of blood vessel injury.

These coagulation factors, which are proteins,with the exception of Calcium and
Thromboplastin, can conveniently be divided into three families: the fibrinogen,
prothrombin, and contact family. The fibrinogen family includes fibrinogen, Factors V,
VIII, and XIII. The prothrombin family includes Factors II, VII, IX, X, Protein C and
Protein S. The contact family of plasma coagulation proteins include: Factor XII or
Hageman factor, Factor XI, Fletcher factor or Prekallikrein (PK), Fitzgerald factor or
High Molecular Weight Kininogen (HMWK) and possibly the Passovoy factor. They are all
involved in the mechanism that generates insoluble fibrin as a final product, by means of
the coagulation cascade. Disorders of secondary hemostasis many times involve a change in
the coagulation proteins. These changes can be a decreased level of a particular factor or
a defect in the way the factor functions.

TOPIC REVIEW

1. T or F. Platelet Factor 3 (PF3) is found in the plasma.

2. Which of the following groups of
factors are involved in the contact phase of coagulation?

Extrinsic PathwayObjectives: 1. Know the role the "extrinsic pathway" plays in hemostasis.2. List the components and the functions of the factors of the "extrinsic
pathway".3. List two major uses of the prothrombin time (PT).

Glossary:

Enzyme:

Organic compound, frequently a protein,
capable of accelerating or producing by catalytic action some change
in a substrate for which it is often specific.

Extrinsic pathway:

Pathway in which fibrin is formed as the
result of the release of tissue thromboplastin into the circulation.

Prothrombin time:

A laboratory coagulation test which measures
the general level of clottability of a plasma sample. It is sensitive
to the factors of the extrinsic clotting system.

INR:

International Normalized Ratio which provides a convenient method for standardizing the
monitoring of Warfarin therapy.

Introduction:
Hemostasis is defined as a property of circulation whereby blood is maintained within a
vessel and the ability of the system to prevent excessive blood loss when injured. One of
the major components needed to provide hemostasis is the coagulation system which involves
the clotting proteins or clotting factors. The coagulation factors, except for
calcium and thromboplastin, are proteins and are involved in a sequential reaction or
coagulation cascade. The last step of the cascade leads to insoluble fibrin as the end
product. The reactions leading to fibrin formation can be divided into the extrinsic,
intrinsic and common pathways. The extrinsic pathway is initiated by the release of
tissue thromboplastin (Factor III) which is exposed to the blood when there is
damage to the blood vessel. Factor VII which is a circulation coagulation factor,
forms a complex with tissue thromboplastin and calcium. This complex rapidly converts Factor
X to the enzyme form Factor Xa.Factor Xacatalyzes
the prothrombin (Factor II) to thrombin (Factor IIa) reaction
which is needed to convert fibrinogen (Factor I) to fibrin. See
figure 3 for "coagulation cascade" diagram depicting the extrinsic,
intrinsic and common pathways. The Prothrombin Time or PT is a laboratory
screening test used to detect coagulation disorders. It measures the activity of the
factors of the extrinsic pathway including factors II, V, VII, X, and
fibrinogen. The extrinsic factors not measured in the PT test are Factors
III (Thromboplastin), and IV (Calcium). The PT is also used to monitor
oral anticoagulant therapy such as warfarin.

Warfarin is a drug used in patient therapy to prevent thrombosis. It inhibits the
synthesis of the vitamin K dependent factors, factors II, VII, IX and X by blocking the
regeneration of vitamin K and shows a dose dependent effect. As more warfarin is ingested
orally, the greater the reduction in the functional levels of vitamin K dependent factors.
See Figure 4 for the effect of warfarin on the synthesis of
clotting factors. Because 3 of the 4 factors affected by warfarin are evaluated by the PT
test, it is commonly used to monitor therapy.

The PT test is performed by adding tissue thromboplastin and calcium to plasma
and measuring the time for clot formation. It can be performed either manually by tilt
tube method or mechanically by use of a fibrometer or a photo-optical instrument. The PT
reagent used in the testing provides the tissue thromboplastin and calcium. The sources of
thromboplastin can be human or rabbit brain, lung, placental, brain/lung combination, or
produced by recombinant technology. The necessary calcium is added to the reagent either
at the time of manufacture or prior to testing.

The PT can be done as either a one-stage or a two-stage assay, although the
one-stage procedure is the most widely used and preferred. Thromboplastin reagent (0.2 ml)
is warmed at 37C then forcibly added to plasma (0.1ml) which also has been heated to 37C
and a timer is started. As soon as the clot forms indicating fibrin formation, the timing
stops and the time is recorded to the nearest tenth of a second. The expected normal range
for a PT is 10-14 seconds depending on the type of reagent used.

Variation in the composition and responsiveness of PT reagents have necessitated
the use for standardization. The International Normalized Ratio or INR was
developed for the purpose of standardizing the monitoring of warfarin therapy.

Several factors may contribute to the differing degrees of responsiveness observed for
various thromboplastin reagents. Some of these include the species and tissue source, the
relative concentrations of other components of the reagent formula etc. The responsiveness
of the thromboplastin reagent needs to be considered to make the PT an effective
way of monitoring warfarin treatment. The responsiveness of a thromboplastin reagent
toward plasma samples from patients receiving warfarin is described by a value called the
International Sensitivity Index (ISI).

The calculation of the INR is obtained by using the following calculation:
The lower the ISI, the more responsive the reagent. The differences in the responsiveness
of thromboplastins to the reduction of clotting factors II, VII and X are responsible for
the difference in dosage of oral anticoagulants.

In summary, defects in the normal hemostatic mechanism can be listed as two types. One
is the failure of any of the processes that lead to the hemostatic plug formation which
may lead to a bleeding disorder and inappropriate activation of the hemostatic mechanism
which may cause thrombosis. Laboratory investigations and determinations are needed to
identify the eXact nature of the underlying bleeding disorder. Screening tests
such as the PT are initially performed. Based on these results, further, more complex
testing may be needed leading to follow-up corrective action and treatment.

TOPIC REVIEW

1.The prothrombin time is a screening test used to evaluate the
_______________ pathway of coagulation.

2.T or F. The PTT test is the most
common method used for monitoring oral anti-coagulant therapy?

3. What is the advantage of a
thromboplastin reagent which has a lower ISI value?

Objectives:1. Define activated partial thromboplastin time (aPTT)2. Describe two major uses of the aPTT

Glossary:

Activated partial thromboplastin time (APTT)

One of the tests used for screening patients
for a bleeding tendency. Specifically, adequate levels of the
coagulation factors XII, XI, IX, VIII, X, V and II must be present for
the test to be normal. The test also serves as the basis for other
test procedures such as certain factor assay tests.

Intrinsic

Originating from within

The intrinsic pathway of Coagulation is activated when circulating Factor XII
comes in contact with and is bound to a negatively charged surface. This causes a change
in the molecular configuration of Factor XII and in concert with HMWK and prekallikrein
it becomes an active enzyme, XIIa. This activated enzyme is then able to
bring about a similar change in Factor XI. After activation, Factor XIa,
in a calcium dependent reaction, converts Factor IX to its active form, Factor
IXa. A phospholipid surface is also needed for Factor IXa
conversion and is provided by activated platelets, as Platelet Factor Three (PF3). Factor
IX can also be activated by the tissue factor, Factor VII complex;
the initiating complex of the extrinsic pathway.Factor Xcan be
activated toFactor Xaby either the Factor
VIIacomplex or by the complex ofFactor IXaandFactor VIII.Factor Xain
the presence ofFactor V, calcium and phospholipid surface
converts Factor II (prothrombin)toFactor IIa
(thrombin)which convertsFactor I (fibrinogen)tofibrin(see Figure 5).

Activated partial thromboplastin time (aPTT)is an assay used
to screen for abnormalities of the intrinsic clotting system. It is also used to monitor
the anticoagulant effect of circulating heparin.

AnaPTTassay is performed by adding to platelet poor plasma a Factor
XII activator, a phospholipid, and calcium ions. Factors I, II, V,
VIII, IX, X, XI, XII, prekallikrein(Fletcher Factor) and high molecular
weight kininogen(HMWK)are measured. An abnormalaPTTresult might indicate the presence of an acquired inhibitor or a deficiency in
any of the coagulation factors exceptFactors VII and XIII.

For in vitro analysis, some commonly used activators are glass, ellagic acid, kaolin,
silica and celite. All of these except glass, are used in aPTT reagents
and serve the same function of activating the clotting mechanism. Phospholipids are
platelet substitutes and accelerate the reactions involved. Sources of phospholipids are
rabbit brain, cephalin (dehydrated rabbit brain), bovine brain, and soy bean.

When adequate levels of all the coagulation factors are present in plasma, the aPTT
test result is normal. Normal ranges of the factors vary from approximately 50-150% of
normal. In general an aPTT reagent should be able to detect factor levels
of 30% or less. If aPTT results are prolonged and there is no indication
of a factor deficiency, an acquired inhibitor may be present.

Heparin will also cause a prolonged aPTT. This commercial product is
prepared from beef lung or porcine intestinal mucosa and is administered via intravenous
or subcutaneous injection. Heparin with its plasma co-factor Antithrombin III,
inhibits coagulation immediately after being administered. It is the drug of choice for
treating venous thrombosis by preventing fibrin formation.

The aPTT, although useful in monitoring high level heparin therapy,
has had variable effectiveness in monitoring low dose heparin therapy and low molecular
weight forms of heparin.

TOPIC REVIEW

1.What screening test is used for
detecting abnormalities in the intrinsic pathway?

Objectives:1. Name the most important inhibitors of Hemostasis.2.Know the function of naturally occurring inhibitors.

Glossary:

Antithrombin III

Natural inhibitor of the coagulation system

Protein C

Natural inhibitor of the coagulation system

Protein S

Protein C co-factor

Once Coagulation is initiated, the body has mechanisms for avoiding massive thrombus
formation. Physiologic balancing of the Hemostatic mechanism to limit uncontrolled
bleeding and clotting is an important aspect in the Hemostatic response. There are a
variety of biological control mechanisms which aid in the control of blood coagulation.
These include the ability of the liver and the reticulo-endothelial system to clear
activated clotting factors from the circulation, the prevention of the high concentrations
of activated factors at a given location within the circulation by a constant blood flow,
and natural inhibitors in the plasma such as Antithrombin III and the Protein
C-S System.

Antithrombin III (AT-III) is the most important inhibitor of the
coagulation enzymes. AT-III binds to activated factors rendering them
inactive (Figure 6). The primary function is to inactivate thrombin. Inactive factors and
cofactors are not neutralized by AT-III, since it only binds to the
enzymatic factors. The process of binding the active forms of the clotting factors (XIIa,
XIa, Xa, IXa) and thrombin to AT-III
is greatly accelerated by heparin to an almost instant neutralization. AT-III
inhibits not only coagulation enzymes but also plasmin and kallikrein.

Patients with decreased AT-III levels are subject to an increased risk
of thromboembolism even in cases of slightly reduced AT-III levels,
therefore the Antithrombin III assay is an important part of a
prethrombotic workup.

Antithrombin III levels are affected by several other disease states.
Individuals suffering from severe hepatic disorders such as cirrhosis or acute hepatitis
have significantly depressed AT-III levels, while disease accompanied by
inflammation may show elevations.

Protein C is an inhibitor of the activated Factors Va
and VIIIa.(See Figure 6) This is its anticoagulant function. Protein
C also inactivates tissue plasminogen activator inhibitor (PAI)
which increases the activity of tissue plasminogen activator (tPA) which
enhances fibrinolytic activity. Therefore, it can be said that Protein C
has both anticoagulant and fibrinolytic functions. Just as Antithrombin III
has a co-factor which is heparin, Protein C has a co-factor which is Protein
S. Both Protein C and Protein S are vitamin K
dependent factors. Enhancement of Protein C anticoagulant functions is
achieved by Protein S. Patients with Protein C and/or Protein
S deficiencies have a thrombotic tendency. Patients also may acquire deficiencies
of Protein C and Protein S with liver disease and
disseminated intravascular coagulation (DIC).

TOPIC REVIEW

1.Name two inhibitors of
Coagulation.

1.

2.

2.What is the function of
an inhibitor?

3.Deficiencies of
Antithrombin III and Protein C result clinically in __________________.

Objectives:1.Define and describe the fibrinolytic system, differentiating between
fibrinogenolysis and fibrinolysis.2.Identify activators of plasminogen.3.Differentiate between FDP and fdp.

Glossary

Fibrinolysis:

Dissolution and localization of a fibrin
clot.

Plasmin:

Active portion of fibrinolytic system: has the ability to dissolve formed fibrin clots;
also has similar effect on other plasma proteins and clotting factors.

The last stage of coagulation isfibrinolysis, which is the
dissolution and localization of a fibrin clot. These functions are carried out by enzymes
and their inhibitors. A disruption or breach of the fine balance of this fibrinolytic
system can result in bleeding or thrombosis.

The components of the fibrinolytic system are schematically shown in Figure 7.Fibrinolysisis mediated by
activation of plasminogen to plasmin. This is accomplished by:

Intrinsic activation (plasma based) initiated through Factor
XIIaandkallikrien.Thus, the contact
system of coagulation serves as an intrinsic activator.

Extrinsic activation(cellular based) initiated by way of
stimuli such as vascular injury, ischemia, exercise, stress and pyrogens.

Activators of plasminogen convert it to the active enzyme plasmin. Plasmin, in turn,
acts to split the fibrin clot into fibrin degradation products. To balance this activity
there are inhibitors. The most important inhibitor of plasminogen activators is PAI-1,
which is fast acting. Alpha2-antiplasmin, another principal inhibitor of
fibrinolysis, inhibits plasmin (See Figure 8).

Soluble fibrinogen is cleaved by thrombin to form fibrin monomers. The fibrin monomers
aggregate to form fibrin polymers, unstable fibrin clots. Thrombin also activates factor
XIII to an activated enzyme, factor XIIIa, which in
the presence of calcium converts fibrin polymers to a stable fibrin clot. Plasmin can
degrade or split both fibrinogen and fibrin into fragments, X, Y, D and E.
Fibrinogen degradation products (FDP) are the products of
fibrinogenolysis and are detected by the FDP assay. Fibrin degradation products (fdp)
are the product of fibrinolysis. The only time D-dimers (cross linked D-domains) are
present is after the degradation of a stable fibrin clot (See Figure
9). These tests (FDP and D-Dimer) will be described in future modules.

There are many conditions that can affect the fibrinolytic system resulting in an
increased or decreased activity of fibrinolysis. Samples of such conditions are
Disseminated Intravascular Coagulation (DIC), trauma from surgical
procedures or accidents, deficiencies in or consumption of the various inhibitors and
activators of the fibrinolytic system.

Continued study of the fibrinolytic system unlocks it's complexities . Always on the
horizons are newer and more sensitive and specific methods of evaluating this system, thus
providing better diagnostic tools.

TOPIC REVIEW

1.Define Fibrinolysis.

2.

TorFPlasmin cleaves fibrin into
small fragments.

TorFThere are three (3) pathways of
activating plasminogen.

TorFD-dimers are fragments only
detected when fibrin clots are degraded.